Enzymatic substrates that become fluorescent after being acted upon by an enzyme generally are well known. Such fluorescent substrates typically have two components that are bound to one another through, for example, a covalent chemical bond. One component is a fluorescent molecule that is capable of fluorescing by first accepting light energy and then emitting light energy. The other component is an entity that prevents the fluorescent molecule from accepting or emitting light energy when the two components are covalently bound to one another. In the presence of an appropriate enzyme, the enzyme cleaves the covalent bond between the two components and separates one component from the other to permit the fluorescent molecule to accept and emit light energy. In other words, the enzyme frees the fluorescent molecule and allows it to fluoresce.
Fluorescent enzyme substrates, such as those described above, have demonstrated utility in the diagnostic industry. For example, fluorescent substrates have been used in diagnostic assays designed to detect disease causing agents such as an HIV analyte in a test sample. Heterogeneous immunoassays are one type of diagnostic assay where fluorescent substrates have been employed. According to such assays, an analyte which may be found in a test sample is separated from the test sample using a solid support material that is capable of specifically binding the analyte. The bound analyte can then be contacted with a conjugate comprising a specific binding member and an appropriate enzyme. The conjugate also specifically binds the analyte and a result, binds the enzyme to the analyte. The support material and any bound enzyme can then be contacted with a fluorescent enzyme substrate that can be cleaved by the enzyme. The enzyme acts upon the substrate to yield the fluorescent product that then can be detected as an indication of the presence of the analyte on the support material, and therefore in the test sample. Many other assay configurations using fluorescent enzyme substrates are well known.
Detecting fluorescence emitted from the fluorescent component of a fluorescent enzyme substrate is typically achieved in two steps. In particular, the fluorescent molecule is first excited with light energy and subsequently, the fluorescence emitted from the fluorescent component is then detected. Generally, fluorescent molecules can be excited with light energy from, for example, a laser or another suitable light source. Fluorescence is detected with a device designed to detect light energy of a wavelength that is emitted by the fluorescent molecule. Such excitation and emission detection systems generally are designed to operate at particular wavelength ranges and can be one of the most expensive components of an assay system.
Many fluorescent substrates and the enzymes that cleave these substrates to release fluorescent molecules are known. Ideally, fluorescent substrates should be soluble and stable in aqueous buffers, should have a high affinity for the enzymes that act upon them, and should yield a strong signal upon enzymatic action. Additionally, it would be advantageous to have a enzymatic substrate with all these properties and have spectral properties that are similar to presently existing fluorescent substrates. The advantage of having such a substrate resides in the fact that such a substrate would enable multiple analytes to be detected in a single assay without the need for additional excitation and detection systems to excite and detect a fluorescent molecule having distinct spectral properties.
Unfortunately, while many fluorescent substrates are known, substrates having all of the properties listed above are are currently believed to be unknown. For example, some substrates may have the desired spectral properties, but may not be sufficiently soluble. Other substrates may be sufficiently soluble but have stability problems such that the original compound breaks down in solution to yield an entirely different compound which may or may not have the spectral properties of the original compound. Still other substrates may not bind well with enzymes which thereby necessitates the use of excess substrate to yield a sufficiently detectable signal. Accordingly, there is a need for a fluorescent enzyme substrate that (i) is soluble and stable in aqueous buffers, (ii) has a high affinity for the enzymes that act upon it, and (iii) yields a strong signal upon enzymatic action. Moreover, there is a need for a fluorescent substrate that exhibits these properties and has spectral properties that are similar to existing substrates.